Biotreatment of organic waste has been used for centuries as a means of modifying ecosystems. Composting, for example, has been used from the dawn of agriculture as a way to produce soil amendments from organic waste and to enhance nutrient cycling. In this century, biotreatment of wastewater has also been used as a way to remove organic matter (U.S. Pat. No. 5,540,840), as well as to facilitate water reclamation through various other means (U.S. Pat. No. 5,792,650).
Most existing wastewater treatment plants were built decades ago at a time when less stringent water quality standards (e.g., those based on criteria like biological oxygen demand (BOD) and chemical oxygen demand (COD)) were in place. Furthermore, these wastewater treatment plants commonly relied on activated sludge processes as a core component of wastewater biotreatment. While treatment systems that utilize only physio-chemical processes are available for water reclamation (U.S. Pat. No. 4,851,131), activated sludge-based systems remain as standards, particularly for aerobic wastewater treatment systems (Hallas, Laurence E. and Michael A. Heitkamp, 1995. “Microbiological treatment of chemical process wastewater.” In: Young, Lily Y. and Carl E. Cerniglia (eds) Microbial Transformation and Degradation of Toxic Organic Chemicals, New York: Wiley-Liss, pp. 349-387).
Though improvements in component unit processes of wastewater biotreatment systems have been made, translating these improvements into consistent realization of higher quality effluents has been hampered by the need to rely on monitoring processes that permit only slow or passive adjustments in control processes. For some narrowly circumscribed situations, however, monitoring technology has progressed to approach real-time control of unit processes in wastewater biotreatment systems. For example, NADH fluorescence and pH signals may be utilized to optimize the rate of carbonaceous nutrient feeding to a anoxic reactor (U.S. Pat. No. 6,106,718), or, as another example, the level of expression by reporter bacteria of a gene encoding a bioluminescent reporter protein may be monitored photometrically as being correlative with the absence or presence of bacteriocidal toxicity in a wastewater treatment stream (U.S. Pat. No. 6,110,661).
In general, technologies for monitoring the abundance and expression of genes encoding proteins that mediate the microbial degradation of target components of wastewater treatment streams have been lacking. Methods that permit informed, active control of wastewater biotreatment systems through the utilization of information derived from such monitoring have also been lacking. The present invention overcomes these and other deficiencies in the prior art and meets a long-felt need for technology to accomplish monitoring of both the abundance and expression of key microbial genes, so as to permit active control of wastewater biotreatment systems.
Wastewater treatment, whether for municipal or industrial wastewater, has generally been pursued as a battery of treatments that may be divided into three general levels: primary, secondary, and tertiary. Primary treatment typically involves the removal of a substantial amount of suspended solids from a wastewater sample. A principle technology for primary treatment is sedimentation. During sedimentation, settleable solids are removed from raw wastewaters. For organic industrial discharges with a low-to-moderate suspended solids content, passage through an equalization basin may be required if the organic matter content or hydraulic flow rate of the wastewater varies appreciably over time.
Secondary treatment may be viewed as generally being bioremedial in nature. It usually consists of bio-oxidizing those organic solids that remain after primary treatment has been completed. Commonly used technologies in the secondary treatment of primary effluent rely on suspended growth biotreatments, and particularly, on activated sludge processes. Although secondary treatment systems and their component unit operations and processes have been the loci of key technological advances in recent decades, limitations in monitoring processes often prevent active control processes from being implemented in many secondary treatment systems.
Typically aerobic secondary, and often tertiary, treatment occurs in biological reactor systems. These fall into two general categories—aerobic suspended growth and attached growth systems. In either case, the principle is the same: to bring microbial biomass in contact with (i) organic compounds as a source of energy, (ii) an electron acceptor (like oxygen or nitrate), and (iii) appropriate nutrients for microbial growth. As organic components are degraded, a means to separate the treated liquor from the increased biomass must be provided.
In aerobic systems, the technology of continuous flow activated sludge has been the mainstay technology of secondary treatment. In a continuous flow activated sludge system, as depicted in FIG. 1, waste is first mixed and aerated with microorganisms in an aeration tank 50 for a defined period of time. A second tank, a clarifier 60, provides for separation (i.e., clarification) of water with a greatly reduced organic (sometimes inorganic) content from biomass that settles into an activated sludge blanket 65.
Despite passage through well-tested secondary treatment processes, aqueous discharges from secondary treatment systems may not meet water quality standards. Levels of suspended solids, nutrients, or specific regulated compounds in such aqueous discharges may be unacceptable. In such cases, tertiary treatment is required. Tertiary treatment options include additional chemical treatment (e.g., activated carbon filtration, ozonation, coagulation, air stripping, and ion exchange processing) and/or biological treatment (e.g., polishing components with specific microbes on activated carbon or alginate).
As might be expected, continuous flow activated sludge processes are mechanically well understood. Key operating variables are used to design and operate systems successfully. For example, initial waste characterization is followed by reactor sampling in order to define operational system organic loadings (food/microorganism or F/M ratios). In addition, rates of air delivery and oxygen transfer are determined and adjusted to achieve desired levels of wastewater processing. Finally, the making of similar determinations and adjustments for rates of clarification, as well as of microorganism growth (and subsequent biomass wastage) are made. Various augmentations of component unit processes may also be used in order to improve continuous flow activated sludge systems. For example, the addition of activated carbon to the aeration tank has provided a successful variation in treating dilute but variable wastewater.
In wastewater biotreatment systems monitoring methods are required to detect and maintain process stream parameters within optimal ranges or, at least, to prevent catastrophic perturbations in the operation of such systems and to avoid excursions beyond water quality limits. In addition to microorganism diversity, the level of constant solids, F/M ratio, mean cell retention time (MCRT), settling rate and sludge volume index, as well as oxygen uptake rate (OUR) and specific oxygen uptake rate, are among the process control parameters measured in wastewater biotreatment systems.
This type of information is helpful in deciding what adjustments should be made, e.g., in channeling return activated sludge flow versus waste activated sludge flow (see, respectively, 67 and 69 of FIG. 1; see also, Wastewater Engineering: Treatment, Disposal, and Reuse (McGraw-Hill Series in Water Resources and Environmental Engineering, 1991) by George Tchobanoglous, Franklin L. Burton, and Metcalf & Eddy, Inc. Staff).
Biological oxygen demand (BOD) is the amount of oxygen that would be consumed if all the organic material in one liter of water were oxidized by microorganisms. In a rudimentary method of measuring five-day BOD (BOD5), two equal volumes of water are sampled from a test pool and each aliquot is diluted with a known volume of distilled water which has been thoroughly shaken to insure oxygen saturation. The concentration of oxygen within one of the aliquots is then measured using an oxygen meter, while the remaining aliquot is sealed and placed in total darkness. Five days later, the concentration of oxygen within the second aliquot is measured using an oxygen meter. BOD5 is determined by subtracting the second meter reading from the first.
The greater the relative amount of organic matter to be oxidized, the greater the relative amount of oxygen that will be needed by microorganisms within a wastewater biotreatment system in order to oxidize that amount of organic matter. Furthermore, BOD5 is heavily influenced by the microorganism seed source and the degree of acclimation of sample microorganisms to waste components to be used as substrates in measuring BOD5. As a result of these relationships, BOD5 can be used to measure wastewater biotreatment efficiencies for specific substrates. For example, formaldehyde in wastewater of certain types may be bio-oxidized by activated sludges of various kinds and thus be detectable as BOD5. However, degradation of glyphosate (i.e., N-phosphonomethylglycine) and N-phosphonomethyliminodiacetic acid (PIA) from the same wastewater may not be detectable as BOD5 if there are no gox-expressing microorganisms in the activated sludge.
Alternatively, oxygen demand can be measured using a chemical oxidizing agent. This is called chemical oxygen demand (COD). COD may be expressed in terms of the milligrams of oxygen required to chemically oxidize the organic contaminants in one liter of wastewater. COD values are generally higher than BOD values. Typical COD values for domestic wastewater range from 200 to 500 mg/L. COD provides an indication of the theoretical oxygen demand and is often used in place of BOD, particularly because COD determinations may be established after only a few hours, while standard BOD5 determinations require five days.
Determining total organic carbon (TOC) levels in wastewater has been used for many years as a method for estimating pollution levels. Several methods may be utilized to measure wastewater TOC, though all methods typically measure the organic carbon content of aqueous samples. Typical TOC values for domestic wastewater range from 100 to 300 mg/L.
Mean cell residence time (MCRT) is the length of time that the average microorganism remains in a treatment process (e.g., a continuous flow activated sludge process) considering the removal of microorganisms (e.g., via the sludge wasting process). MCRT may also be expressed in terms of solids retention time or sludge age. For example, if five days on average are required for the removal of an amount of sludge equal to that typically held in a system, sludge microorganisms will remain in the sludge contained in the system for an average of five days. Accordingly, the sludge age for the treatment process would be five days. For a typical plant through which domestic wastewater is treated by a conventional activated sludge process, a typical range for MCRT would be five to fifteen days. For a plant handling industrial wastewater, a typical range would be 30 to 100 days.
Microorganism diversity profiles in biotreatment systems change with environmental conditions, including dissolved oxygen concentration and hydraulic residence times (HRTs). HRT refers to the average time an aqueous or fluid phase of a biotreatment system remains within the system. MCRT and HRT may differ markedly for some systems, e.g., those utilizing immobilized bacteria technology. Complete mix activated sludge systems provide another example of MCRT and HRT differences. It is common for industrial biological systems employing complete mix activated sludge systems to possess a HRT of 2-7 days, but a MCRT of 30-80 days.
In most systems, however, as HRT increases, more substrate organic matter is sorbed (i.e., both absorbed and adsorbed) as well as biologically degraded by microorganisms. Consequently, the ratio of substrate organic matter to microorganism biomass (i.e., the F/M ratio) decreases, which can dramatically change the structure of the microorganism community.
Mixed liquor volatile suspended solids (MLVSS) includes living and nonliving organic matter and represents a crude approximation of the amount of biomass. Mixed liquor suspended solids (MLSS) also includes inorganic solids and thus is a more crude estimate of biomass. Typically, MLVSS is 70 to 80% of MLSS.
The F/M ratio is also important in maintaining a biotreatment system. Since BOD (or COD) times influent (or exfluent) flow rates is an estimate of the numerator F, and MLVSS is an estimate of the denominator M, BOD (or COD) times influent (or effluent) flow rate per unit of MLVSS provides an estimate of the F/M ratio.
Although these parameters have been successfully used to monitor biological waste treatments systems, they suffer disadvantages. In particular, the measurement of MLVSS is only an approximation of total biological content and further includes all of the organisms found in an activated sludge. These are usually quite diverse and may include bacteria, protozoa, rotifers, fungi, and nematodes, as well as algae and insect larvae. In addition, the MLVSS measurements do not distinguish between living versus nonliving microorganisms.
The primary way that microbial activity has been monitored for a given MLVSS value is by conducting non-specific oxygen uptake rate (OUR) analyses. However, such analyses reflect degradative microbial activity for multiple components of a wastewater sample as opposed to a single component, such as glyphosate. Oxygen uptake rates are also greatly influenced by the presence or absence of more readily degradable components in a wastewater sample.
The dynamics of changes in microbial community structure and the performance of wastewater biotreatment systems are closely related. Thus, an efficient means of accurately quantifying those organisms responsible for specific biological processes would be of considerable benefit in water reclamation because it would allow finer monitoring and control of the wastewater biotreatment systems. It is particularly desirable to monitor the structure of bacterial and other microorganism communities in a way that does not rely on cell culture methods in order to determine not only the type of microorganisms responsible for the degradation of a specific regulated compounds, but also their abundance and activity.
PCR-based methods (U.S. Pat. No. 4,683,202 and US patents citing same) have been useful for detecting genes from microorganisms involved in the degradation of xenobiotic compounds. For example, DNA extraction followed by PCR has been used to detect genes of microorganisms important in the degradation of polychlorinated biphenyl organics (Erb et al., 1993, Appl. Environ. Microbiol. 59: 4065-4073) and naphthalene (Herrick et al., 1993, Appl. Environ. Microbiol. 69: 687-694) in polluted sediments, as well as other genes, including catechol 2,3-dioxygenase genes (Joshi and Walia, 1996, FEMS Microbiol. Ecol. 19: 5-15) and metapyrocatechase homologous genes (Joshi and Walia, 1996, J. Microbiol. Methods 27: 121-128) in petroleum hydrocarbon contaminated groundwater.
PCR-based methods have been used not only to detect, but also to quantitatively estimate, soil bacteria degrading 4-chlorobiphenyl organics (Ducrocq et al., 1999, Appl. Soil. Ecol. 12: 15-27) as well as an uncultured bacterial strain (Lee et al., 1996, Appl. Environ. Microbiol. 62: 3787-3793). Methods of quantitative PCR (qPCR), particularly competitive qPCR (U.S. Pat. No. 5,213,961 and US patents citing same), have been used to follow fluctuations in the diversity of bacterial populations in phenol-acclimated activated sludge (Watanabe et al., 1999, Appl. Environ. Microbiol. 65: 2813-2819). Mesarch et al. (2000, Appl. Environ. Microbiol. 66: 678-683) developed primers specific for catechol 2,3-dioxygenase genes of certain strains of Pseudomonas bacteria so that competitive qPCR could be used in direct, non-cultivation-based techniques for enumerating microbial populations in soil samples.
Watanabe et al. (2000, Appl. Environ. Microbiol. 66:3905-3910) also used qPCR-based methods to estimate the densities of Ralstonia eutropha E2 bacteria present in activated sludge. In addition to estimating densities of R. eutropha E2 bacteria in activated sludge using qPCR with pox gene-specific primers, Watanabe et al. (2000) also used methods of reverse transcription PCR (RT-PCR) to detect, but not quantify, the expression of the pox gene by these bacteria in activated sludge. Through pox gene expression, R. eutropha E2 bacteria are capable of growing on media containing phenol as a sole carbon source. Watanabe designed and used the same pox gene-specific DNA primers for both qPCR and nonquantitative RT-PCR. Dionisi et al. (2002) recently used qPCR-based methods to enumerate ammonia- and nitrite-oxidizing bacteria from municipal and industrial activated sludge (Dionisi, H. M. et al., 2000 Appl and Environ Microbiol, Vol. 68 (1): 245-253). However, no attempt was made to determine the activity of the specific nitrifying bacterial populations.
While PCR-based methods have been shown to have utility for characterizing genes expressed in the activated sludge of wastewater biotreatment systems, monitoring methods are needed to quantify with accuracy both the relative abundance and the relative expression levels of these genes. Furthermore, operational procedures to translate both abundance and expression values derived from the monitoring methods into optimal control parameters are needed for wastewater biotreatment systems. In other words, methods of wastewater characterization that allow accurate monitoring in a near real-time manner for both the abundance and expression of indicator/effector gene combinations are needed, as are methods for using such abundance and expression determinations in order actively to control wastewater biotreatment systems for optimal water reclamation. The need for such methods (and compositions therefor) has become more pronounced with each passing year.